A Two-Heads-Bound State Drives KIF1A Superprocessivity.

KIF1A, a neuron-specific Kinesin-3 motor, is indispensable for long-distance axonal transport and nuclear migration, processes vital for neuronal function. Using MINFLUX tracking, we reveal that KIF1A predominantly adopts a two-heads-bound state, even under ATP-limiting conditions, challenging prior models proposing a one-head-bound rate-limiting step. This two-heads-bound conformation, stabilized by interactions between the positively charged K-loop and negatively charged tubulin tails, enhances microtubule affinity and minimizes detachment. The shorter neck linker facilitates inter-head tension, keeping the heads out of phase and enabling highly coordinated stepping. In contrast, Kinesin-1 (KIF5B) transitions to a one-head-bound state under similar conditions, limiting its processivity. Perturbing KIF1A's mechanochemical cycle by prolonging its one-head-bound state significantly reduces processivity, underscoring the critical role of the two-heads-bound state in motility. These findings establish a mechanistic framework for understanding KIF1A's adaptations for neuronal transport and dysfunction in neurological diseases.